The overall objective of this research proposal is to delineate the biological functions of Twist in promoting cancer initiating cells and metastasis in the pathogenesis of breast cancer. In support for this objective, we had earlier demonstrated that over-expression of Twist, a basic helix-loop-helix transcription factor, in breast cells (MCF-7) promotes epithelial-mesenchymal transition, enhanced tumorigenesis, increased genetic instability, and increased vasculature and permeability within the tumor microenvironment of orthotopic xenografts. We also demonstrated that the over-expression of Twist in breast cells can promote breast cancer initiating cell phenotype characterized by high CD44 expression, little or no CD24 expression and increased ALDH activity. In addition, Twist over-expressing cells exhibit high efflux of Hoechst 33342 and Rhodamine 123 as a result of increased expression of ABCC1 (MRP1) transporters, a property of cancer initiating cells. Importantly, we show that inoculums of only twenty cells of the Twist over-expressing CD44?/low sub-population are capable of forming tumors in the mammary fat pad of SCID mice. In our recent studies, a direct association between Twist expression and the dysregulation of Mucin 1 (MUC1) was demonstrated in breast cancer cells. Interestingly, our studies have also revealed that Twist expression regulates the expression of p27 and choline kinase, which are well-established markers of aggressive cancer phenotypes. In this revised application, we aim to establish the mechanistic basis for the role of Twist in pathogenesis of breast cancer and in altering metabolomics during the ontogeny of breast cancer. Our hypothesis is that Twist expression alters normal cellular pathways, facilitating the differentiation towards myoepithelial progenitor cells and increasing metastatic potential. To test this hypothesis, we propose three specific aims. In Aim 1, we will identify cancer initiating cells induced by Twist and functionally demonstrate their tumor forming potential. In Aim 2, we will determine the molecular mechanisms of Twist induced breast cancer progression and metastasis through its regulation of p27 and choline kinase. In addition, we will longitudinally determine the metabolic changes induced by Twist expression in preclinical models. In Aim 3, we will identify early markers of aggressive phenotypes and cancer initiating markers induced by Twist expression in pure DCIS samples and utilize the samples from the rapid autopsy program to determine if Twist expression can be associated with organ specific breast cancer metastasis. Importantly, we will identify breast cancer initiating cells in patient breast tumors and determine their functional implications in breast tumorigenesis. Taken together these studies will establish the role of Twist in inducing breast cancer initiating cells and metastasis as well as its utility as an early marker of breast cancer. PUBLIC HEALTH RELEVANCE: The project will help define the role of Twist in inducing cancer initiating cells and in metastasis during the pathogenesis of breast cancer. The clinical translatability of these findings will be confirmed in human breast cancer tissue samples such as pure DCIS and that obtained from the rapid autopsy program at Johns Hopkins. We anticipate that the clinical applications derived from the insights and knowledge provided by the successful completion of this research project will lead to molecular approaches to identify and target this lethal subpopulation of cells, which will improve treatment outcomes. In the long term, these studies will help characterize breast cancer initiating cells and provide insights that will contribute to the development of novel imaging markers and chemotherapeutic drugs for breast cancer.